Renewable and Sustainable Energy Reviews 34 (2014) 491500

Contents lists available at ScienceDirect

Renewable and Sustainable Energy Reviews

journal homepage: www.elsevier.com/locate/rser

Process characteristics, inhibition factors and methane yields

of anaerobic digestion process, with particular focus
on microalgal biomass fermentation
Ewa Kwietniewska n, Jerzy Tys
Institute of Agrophysics, Polish Academy of Sciences, 4 Doswiadczalna Street, 20-290 Lublin, Poland

art ic l e i nf o

a b s t r a c t

Article history:
Received 11 April 2013
Received in revised form
3 March 2014
Accepted 15 March 2014
Available online 3 April 2014

World's energy demand has been constantly increasing for decades. Yet, fossil fuels stock, which are used
the most extensively nowadays, may be depleted soon. Moreover, combustion of conventional fuels
leads to excessive carbon dioxide emission. This process causes multitude of unfavorable consequences
for Earth's climate and biosphere. That is why the alternative fuel sources are searched and studied for
years. One of the possibilities of obtaining renewable energy is biogas production from biomass through
anaerobic digestion process. Anaerobic digestion is widely applied to treat various wastes and higher
plants biomass. Laboratory-scale studies proved that microalgal biomass is also a feasible source of
substrate for methane fermentation process. Microalgae are predominantly single cell photoautotrophic
organisms that have the ability to proliferate rapidly and absorb signicant amounts of carbon dioxide at
the same time. This characteristics indicate that these microorganisms can be an efcient source of
biomass for biogas acquiring. Current study constitutes a comprehensive review which compares and
summarizes studies concerning anaerobic digestion of microalgal biomass, specic factors and potential
inhibitors that inuence the process as well as it presents results of empirical studies. This review is
based on the latest publications as well as on older esteemed literature.
& 2014 Elsevier Ltd. All rights reserved.

Keywords:
Microalgae
Biomass
Biogas
Methane
Anaerobic digestion

Contents
1.
2.

3.

4.
5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
Anaerobic digestion of microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
2.1.
Substrate composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
2.1.1.
Theoretical biogas potential and modeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
2.2.
Process temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
2.3.
Water content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
2.4.
4 pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
2.5.
C/N ratio. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
2.6.
Organic loading rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
2.7.
Retention time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
2.8.
Inoculum to substrate ratio. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
Inhibition of the process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
3.1.
Ammonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
3.2.
Sulfur . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
3.3.
Macroelements, microelements, heavy metals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
3.4.
Fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
3.5.
Cell wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
3.5.1.
Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
Co-digestion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
Anaerobic digestion of algal remnants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498

Corresponding author. Tel.: 488 174 450 61.

E-mail address: e.kwietniewska@ipan.lublin.pl (E. Kwietniewska).

http://dx.doi.org/10.1016/j.rser.2014.03.041
1364-0321/& 2014 Elsevier Ltd. All rights reserved.

492

6. Future perspectives .
7. Conclusions . . . . . . .
Acknowledgements . . . . .
References . . . . . . . . . . . .

E. Kwietniewska, J. Tys / Renewable and Sustainable Energy Reviews 34 (2014) 491500

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

1. Introduction
According to International Energy Outlook 2011 [1], total world
energy use rises from 505 quadrillion British thermal units (Btu) in
2008 to 619 quadrillion Btu in 2020 and 770 quadrillion Btu in
2035 (by 53%). To reduce dependence on fossil fuels and to reduce
climate change, there is a need to make a switch to alternative,
renewable energy system [2]. Among renewable energy sources
like sun, wind, hydroelectric and nuclear energy, biomass is a very
promising source of bioenergy. Energy from biomass is regarded as
one of the most important future renewable energy sources,
because it can provide a continuous power generation and it plays
an important role in the current CO2-mitigation policy [3]. Several
biological processes to convert biomass to energy, and thus
provide a source of biofuel, have been studied in recent years.
One of the most important processes of biomass conversion is the
anaerobic digestion (methane fermentation) of organic matter to
produce biogas, consisting mainly of methane and carbon dioxide
[4]. This is the process of biochemical conversion of organic matter
into biogas as a product of the metabolic action of methanogenic
bacteria [35]. When biogas is used to generate energy, it is
possible to generate from 20 to near to 300 kW h of net energy
per tonne of waste [6]. Many calculations have shown that the
capture of CO2 and energy recovery from biogas may considerably
contribute to greenhouse gases (GHG) emission reductions [7] and
at the same time it does not contribute to ozone depletion or acid
rain formation [8]. Another reason for utilizing biomass to generate biogas is that the solid waste product from anaerobic
degradation (digestate) contains remineralised nitrogen and phosphorus and thus can be used as organic fertilizer in terms of its
availability to plants [4,9]. In recent years, research into the
potential biomass feedstock for methane fermentation from different sources has been conducted [10]. Anaerobic digestion
technologies show great adaptability to a broad spectrum of
different input materials. Organic fraction of municipal solid
waste, agricultural and animal wastes, sewage sludge and other
sources could be used [7]. Currently, scientists all over the world
have been examining the possibilities of using microalgae as a
source of biogas for energy applications [10]. In Table 1 a quick
overview and comparison of methane yields obtained from different types of biomass and microalgae are included. The aim of
the current study is to present anaerobic digestion process
general characteristics and inhibitors and confront it with the
possibility of the anoxic digestion of microalgal biomass. The study
Table 1
Comparison of methane yields obtained through anaerobic digestion of various
substrates and microalgae (adapted from [8] and modied).
Substrate for biogas production

Methane yield (m3 kg  1 VS)

Municipal solid waste

Fruit and vegetable wastes
Jatropha oil seed cake
Swine manure
Maize silage and straw
Microalgaea
Lignin-rich organic waste

0.200.53
0.42
0.42
0.34
0.31
0.26
0.20

a
Methane yield from microalgae is calculated as a mean from data gathered in
Table 5; data regard only to digestion at circa 35 1C and during HRT of 30 days.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

.
.
.
.

498
499
499
499

summarizes and compares the results of empirical studies of

biogas productivity from the sole biomass as well as from microalgal post-extraction remains and biomass co-digestion with other
substrates.

2. Anaerobic digestion of microalgae

Anaerobic digestion is the process of decomposition of organic
matter by a microbial consortium in an oxygen-free environment
[9]. Anaerobic digestion involves a series of metabolic reactions
such as hydrolysis, acidication, acetogenesis and methanogenesis
which are conducted by various groups of microorganisms [8]. The
rst group of microorganisms enzymatically hydrolyze complex
organic compounds to monomers (e.g. glucose, amino acids),
which are subsequently converted to higher volatile fatty acids
(VFA), H2 and acetic acid. Then, the acetogens convert higher
volatile fatty acids e.g., propionic and butyric acid, produced, to H2,
CO2, and acetic acid. Eventually, methanogenic bacteria convert H2,
CO2, and acetate to CH4 and CO2 [11]. Typical composition of
biogas produced is shown in Table 2.
Anaerobic degradation of phytoplanktonic cells is a process
which occurs in natural water reservoirs. When algal cells sink
towards the anoxic and aphotic zones of the reservoir, they
eventually die and become a part of bottom deposits. The algal
debris then undergoes fermentation. It leads to ammonium and
phosphate but simultaneously toxic substances release, such as
H2S. This process can deplete stream oxygen reserves at night.
Such depletion is harmful to sh and other wild life in the stream,
and therefore must be avoided [12,13]. The total estimated
biogenic CH4 emission to the atmosphere is approximately 525
715  106 t per year and this rate is rising by about 1% per year
[14,15].
This potential can be exploited for the production of chemical
energy of methane through the fully-controlled combined algalbacterial anaerobic digestion process. Microalgal biomass production is the rst step of the process [16]. The algal biomass
production potential and varied algae cultivation methods are
widely reported in literature [10,1719] and thus this subject will
not be covered in the current study. Next, the biomass is used as
nutrient (as a batch) for feeding the anaerobic bacteria for the
production of methane [16].
Table 2
Composition of biogas generated by anaerobic digestion [6].
Compound

Biogas concentration (%)

CO2
Methane
Water
O2
N2
H2
H2S
Ammonia
Mercaptane
Low molecular fatty acids
Higher molecular substances

2550
5075
66.5
0.91.1
3.94.1
o 0.10.8
o 0.11
In spores
Traces

E. Kwietniewska, J. Tys / Renewable and Sustainable Energy Reviews 34 (2014) 491500

Table 3
Effect of low nitrogen growth conditions on the composition of three Chlorella
species and estimation of the theoretical methane potential (in brackets: computed
theoretical methane potential of the residual biomass after lipid extraction) [13,24],
modied.

Chlorella vulgaris
(control)
Chlorella vulgaris
(low N)
Chlorella emersonii
(control)
Chlorella emersonii
(low N)
Chlorella
protothecoides
(control)
Chlorella
protothecoides
(low N)

Protein
(%)

Carbohydrate
(%)

Lipid
(%)

Caloric
value
(kJ g  1)

CH4 (L CH4
g VS  1)

29

51

18

18

0.64 (0.56)

55

40

23

0.69 (0.48)

32

41

29

21

0.74 (0.62)

28

11

63

29

0.92 (0.76)

38

52

11

19

0.65 (0.60)

36

41

23

24

0.71 (0.62)

493

in Table 3. In regard to elemental composition, cells consist of 50%

of carbon, 110% of nitrogen and less than 1% of phosphorus [25].
For example, in the study of Golueke et al. [12], microalgae
harvested from natural lagoons, consisting principally of Scenedesmus spp. and Chlorella spp., had total nitrogen content from 6 to
8% of which 3% was in the form of ammonia.
2.1.1. Theoretical biogas potential and modeling
Methane production is directly related to organic degradation
[26]. When organic material is degraded anaerobically, the end
result is carbon in its most oxidized form (CO2) and its most
reduced form (CH4). Hence, anaerobic digestion is the most
complete of all fermentation processes because methane cannot
be further reduced and carbon dioxide cannot be further oxidized.
The ratio between CH4 and CO2 depends on the oxidation state of
the carbon present in the organic material, thus the more reduced
the organic carbon content is, the more CH4 will be produced [27].
For a compound CaHbOcNd the anaerobic digestion process can be
written as Eq. (1) [13,27,28]:
Ca Hb Oc Nd 1=44a  b  2c 3dH 2 O-1=84a  b 2c 3dCO2
1=84a b  2c 3dCH4 dNH3
1

Table 4
Specic methane yield for three types of organic compounds [13,27,29], modied.
Substrate

Composition

g COD  g-VS  1

L CH4  g-VS  1

CH4 (%)

Proteins
Lipids
Carbohydrates

C5H7NO2
C57H104O6
(C6H10O5)n

1.42
2.90
1.19

0.4460.496
1.014
0.415

50
70
50

Cultivation of algal biomass requires large amounts of fertilizers and also the waste management of residual biomass (e.g. after
lipid or high-value products extraction) has to be considered.
Anaerobic digestion can solve these issues and balance the
economic and energetic incomes and expenses. Nitrogen and
phosphorus remineralisation occurs during the process [13], so
the residuals remaining after anaerobic digestion could either be
recycled as nutrients for algal cultivation or could be sold as soil
fertilizers and conditioners [20]. Moreover, digestion of microalgal
biomass releases low amount of hydrogen sulphide in comparison
to other types of substrates because of only minimal amount of
sulphurated amino acids in their proteins [13].
There were some experiments on anaerobic decomposition of
microalgal biomass conducted. On the basis of these studies, the
overall view of process characteristics and potential inhibitiors
jeopardizing the process are described below.
2.1. Substrate composition
Chemical, physical as well as biological properties of produced
algal biomass are dependent on cultivation conditions under
which it is grown and simultaneously they determine the anaerobic process run. These properties can be indirectly inuenced by
inducing either optimal and/or stress cultivation conditions e.g.
nitrogen, silicon and phosphate depletion stress, modied light
intensity, high salinity and other [21,22].
Cell composition is strongly species dependent but it is also
deeply affected by environmental conditions [13]. Species classied under Chlorophyceae, Volvocales (Chlorella sp., Botryococcus
braunii, Dunaliella salina) exhibit typical biochemical composition
of 3050% proteins, 2040% carbohydrates and 815% lipids.
Under stress conditions these species are able to accumulate up
to 80% of fatty acids, 80% of hydrocarbons, and 40% of glycerol [23].
Changes in the biochemical composition of cells exposed to
optimal conditions and nitrogen deciency stress are presented

However, it has to be emphasized that this equation does not

take cell metabolism into account. If the composition of the
organic material is known and provided that all the material is
converted to biogas, the theoretical methane yield potential can be
calculated from the Buswell's Eq. (2) which assesses the specic
methane yield in standard temperature and pressure (STP) and is
usually expressed as STP l-CH4 g-VS  1 (volatile solids) [27]:




a b
 c 22:4
lCH4
STP
2
Bo;th 2 8 4
12a b 16c
gVS
where 22.4 is the volume of 1 mol of gas (methane) at STP
conditions [27]. Table 4 presents methane yield in relation to
various types of organic compounds.
Theoretical methane yield might be also calculated from the
chemical oxygen demand (COD) of the substrate. The COD test
measures the organic matter concentration by measuring the
oxidant (e.g. dichromate) consumption for the oxidation of the
organic material in aerobic conditions [28]. Organic matter consisting of only C, H, and O is theoretically fully oxidized to CO2 and
H2O. The theoretical COD value of a specic compound can be
calculated from stoichiometric considerations. If this theoretical
value corresponds to the experimental value, it could be concluded
that the oxidation of the organic material is complete [27]. Biogas
production in relation to COD is about 0.5 l g  1 COD removed,
corresponding to a methane production of approximately 0.35 l
CH4 per g of COD removed (3) [27,30].


lCH4
CODsubstrate  0:35 STP
gCODsubstrate
theoreticalmethaneyieldlCH4 g VS  1
CH4 1

and free energy release

The COD of methane is 4 mg mg
from the oxidation of methane per 1 g COD is 12.52 kJ [28]. COD is
also a good indicator of the progress of the degradation during
process, as any undigested material will require oxygen (in an
aerobic environment) to complete degradation [9].
In order to predict and understand anaerobic digestion better,
mathematical modeling of the process has been developed. The
rst simple models date back to the mid-sixties [31]. Nevertheless,
as the knowledge of methane fermentation has increased, more
complex and realistic models have been evolved. They have
included more and more specic reactions of anaerobic digestion
process and a range of factors which can inuence digestion
efciency [32]. Further model development has addressed adaptation the digestion to specic types of substrates e.g. manure,

494

E. Kwietniewska, J. Tys / Renewable and Sustainable Energy Reviews 34 (2014) 491500

sludge, wastewater [31]. Currently, the most approved and comprehensive, generalized model of anaerobic digestion is ADM1
(Anaerobic Digestion Model No 1) which was developed by The
IWA Anaerobic Digestion Modelling Task Group, and the report
concerning its work was published by Batstone et al. [33,34]. The
structured model includes multiple steps describing biochemical
(19 reactions) as well as physicochemical processes. It is assumed
that the latter are independent of microorganisms. The model is
based on degradable COD as a substrate [33,34]. Detailed description of the variables, equations and kinetics as well as structured
model of the reaction paths are included in Batstone et al. [33,34].
There were few studies concerning tting this model to
anaerobic digestion of microalgae [35]. The authors have adjusted
the model to suit microalgal process best. As the hydrolysis is a
limiting step, they have used Contois model for it, which assumes
that the kinetics do not depend on the substrate concentration,
but on the amount of substrate per biomass unit. The authors
conclude that this submitted model ts very well the data
provided by a 140 day experiment of Chlorella vulgaris digestion
[36].
2.2. Process temperature
There are different fermentation techniques applied, which are
distinguished mainly on the basis of the operating temperature
[6]. Three temperature regimes can be used in anaerobic digesters:
psychrophilic, mesophilic, and thermophilic with varied optimum
temperature ranges for the domination of different strains of
methane-forming bacteria [37]. Psychrophilic fermentors operate
at about 25 1C, mesophilic ones at around 35 1C and thermophilic
at around 55 1C. Generally, an increased temperature has a positive
effect on the metabolic rate of the microorganisms and the process
runs faster, but the thermophilic process is harder to control and
needs more energy to keep the fermenter at the xed temperature
[7,38]. At 55 1C more rapid degradation of fatty acids was found
than at 38 1C as well as retention time was shorter as 95% of the
methane yield was obtained after 11 days under thermophilic
conditions compared to 27 days under mesophilic conditions [9].
Chae et al. [39] reported that biogas production rate was reduced
due to even small changes in temperature and Appels et al. [40]
claims that the process can fail at temperature uctuations of even
1 1C per day. Ehimen et al. [41] reported an increase of 5461% in
CH4 yield from algal remnants when temperature of the process
was increased from 25 to 35 1C.

biogas yield in anaerobic digestion is 6.87.2 [37]. Methanogenic

bacteria are extremely sensitive to pH uctuations and prefer pH
around 7.0 as the growth rate of methanogens is greatly reduced
below pH 6.6 [9,37]. Acid-forming bacteria are less sensitive and
tolerate pH in range of 4.08.5, but the optimal pH for hydrolysis
and acidogenesis is between 5.5 and 6.5 [9,37,40]. Therefore, some
designers prefer the isolation of the hydrolysis/acidication and
acetogenesis/methanogenesis processes in two separate stages [9].
At the beginning of the fermentation, acidogens and acetogens
produce acids and CO2 and, as a consequence, pH decreases.
Afterwards, the methane-producing bacteria consume the acids,
the pH of the digester increases and then stabilizes [37]. This factor
is also important because it inuences the proportion of ionized
and non-ionized forms of inhibitors of methanogenesis. Excessive
fatty acids, hydrogen sulphide, and ammonia are toxic only in their
non-ionised forms (FA and H2SpH below 7, NH3pH above 7) [9].
2.5. C/N ratio
The factor that is inseparable with pH is carbon to nitrogen
ratio (C/N ratio). The optimal C/N ratio for anaerobic degradation
of organic waste is 2035. When the C/N ratio is lower, it means
that the material is protein (and nitrogen) rich. Anaerobic digestion of such material results in increased content of free ammonia
released [8]. For some species of algae the high proportion in
proteins is characterized by a low C/N ratio if compared with
terrestrial plants. Carbon to nitrogen ratio of freshwater microalgae on average is 10.2 while terrestrial plants has this ratio of
about 36 [13]. In empirical studies, authors report that e.g. carbon
to nitrogen ratio of Spirulina maxima biomass was very low 4.2
[44], algae from natural reservoir (mainly Chlorella sp. and Scenedesmus sp.) 6.7 [45], Chlorella post-extraction residues 5.4 [41]
and similar (Table 5). It was found that fermentation of such
substrates releases great amount of ammonia (7000 mg l  1) as
well as large amount of volatile fatty acids was observed at the
same time, as a consequence of the toxic effect of ammonia on the
anaerobic bacteria [13]. This factor greatly inuences pH uctuations due to ammonia release which increases the pH and
inhibition that results with VFA content increase and pH lowering
at the same time.
The two-stage reactor with biomass retention has been
reported to be considered the only type capable of reliable activity
with C/N ratios less than 20 [9]. To overcome this obstacle and
increase C/N ratio, co-digestion with low-proteinaceous substrate
in appropriate ratio is applied.

2.3. Water content

2.6. Organic loading rate
Anaerobic plants can also be classied on the basis of water
content of the batch. Dry systems operate with 3040% dry matter
in the feedstock, and wet systems operate with 1025% dry matter
[7]. It has been reported that the highest methane production
rates occur at 6080% of humidity [8]. However, biomass sources
containing much water (even containing less than 40% dry matter)
can be processed without any pretreatment [3,9].
Fresh algal slurry after harvesting contains only 210% of dry
mass [12,42,43]. Thus, the costs of downstream processing in e.g.
biodiesel production is very high because of the high energy
consumption for drying, extraction and conversion processes.
The use of anaerobic digestion technology eliminates these obstacles because the downstream processes of the algal slurry are
minimal [20].
2.4. 4 pH
The next factor which inuences the fermentation process is
pH. As a rule, the most favorable range of pH to achieve maximal

The organic loading rate (OLR) is the amount of volatile solids

to be fed into the digester each day in continuous process. As the
organic loading rate increases, the biogas yield increases to some
extend but above the optimal OLR the volatile solids degradation
and biogas yield decreases due to overloading [46,47]. On rst
days of the fermentation, the addition of a large volume of new
material each day may cause a signicant change in digester's
environment and temporarily inhibit bacterial activity [12]. It
happens due to the fact that hydrolysis/acidication bacteria can
produce to much volatile fatty acids from excessive substrate in a
short time. As a consequence, it lowers the pH of the digester and
methane-forming bacteria are not able to convert so much acids to
methane. For example, in study of Ehimen et al. [41] the most
effective organic loading rate was 5 g VS l  1. At higher loading
rates an increase in the valeric and butyric acids content occurred,
which resulted in inhibition. De Schamphelaire and Verstraete
[48] received higher biogas yields when loading rate was the
lowest (0.6 g VS l  1 AD sludge).

E. Kwietniewska, J. Tys / Renewable and Sustainable Energy Reviews 34 (2014) 491500

495

Table 5
Summary of the results of empirical studies of anaerobic digestion of sole and whole microalgal biomass.
Species

C/N

Temp.
[1C]

Methane yield [m3 kg  1 VS

introduced]

HRT

References

Scenedesmus spp., Chlorella spp., mixed, harvested from natural

lagoon
Scenedesmus spp., Chlorella spp., mixed, harvested from natural
lagoon
Spirulina maxima
Nondened mixed culture dominated by Chlorella

35

0.31

30

Golueke et al. [12]

50

0.32

30

Golueke et al. [12]

4.2

35
34

20
14
25
45

Samson and LeDuy [44]

De Schamphelaire and Verstraete
[48]

Nondened mixed culture dominated by Chlorella

41

14
25

De Schamphelaire and Verstraete

[48]

Scenedesmus spp. and

Non-axenic culture of
Non-axenic culture of
Non-axenic culture of

6.7

35
33
33
33
54
33
54
35
35
38
38
38
38
38
38
35
35
30
37
37

0.31
0.35
0.44
0.60
(biogas containing 4065% methane)
0.280.35
0.390.47
(biogas containing 4065% methane)
0.100.14 (573 ml l  1 day  1)
0.21
0.35
0.13
0.17
0.27
0.29
0.24
0.147
0.293
0.387
0.218
0.323
0.325
0.178
0.201
0.14
9291294 ml of biogas
0.286
0.024

10
30
30
2.2

Yen and Brune

Zamalloa et al.
Zamalloa et al.
Zamalloa et al.

Chlorella spp.
Scenedesmus obliquus
Phaeodactylum tricornutum
Scenedesmus obliquus

Non-axenic culture of Phaeodactylum tricornutum

Chlorella vulgaris
Chlorella vulgaris
Arthrospira platensis
Chlamydomonas reinhardtii
Chlorella kessleri
Dunaliella salina
Euglena gracilis
Scenedesmus obliquus
Microcystis sp. from Taihu lake
Microcystis sp. from Taihu lake
Species unknown
Chlorella vulgaris
Dunaliella tertiolecta

6
6

2.2
28
16
32
32
32
32
32
32
30
30
28
49
49

[45]
[64]
[64]
[64]

Zamalloa et al. [64]

Ras et al. [36]
Ras et al. [36]
Mussgnug et al. [62]
Mussgnug et al. [62]
Mussgnug et al. [62]
Mussgnug et al. [62]
Mussgnug et al. [62]
Mussgnug et al. [62]
Zhong et al. [76]
Zeng et al. [53]
Salerno et al. [54]
Lakaniemi et al. [61]
Lakaniemi et al. [61]

2.7. Retention time

2.8. Inoculum to substrate ratio

Retention time is the time required to degrade the organic

matter completely and it is connected with microbial growth rate.
The retention time depends on process temperature and batch
composition. The average retention time for waste treated in a
mesophilic plant is 1530 days and a bit shorter for thermophilic
plant [49]. There are two signicant types of retention time the
solid retention time (SRT) the average time the bacteria (solids)
are in the anaerobic digester, and hydraulic retention time (HRT).
HRT is dened by following Eq. (4) [50]:

In case of batch process, Zeng et al. [53] investigated inoculum

to substrate ratio (ISR) (based on volatile solids) as a key factor of
methane productivity from algal biomass. It was reported, that
cumulative methane yield was the highest when ISR was 2.0 while
the percentage of methane in biogas volume was increased up to
45% as the ISR decreased to 0.5.

3. Inhibition of the process

HRT

V
Q

V is the volume of the biological reactor and Q the inuent ow

rate in time
Digestion time is one of the main factors inuencing the CH4
yield [41]. Effective hydraulic retention time depends on the type
of substrate and on loading rate, and reaches up to a couple of
weeks. Shorter HRT usually results in accumulation of VFA,
whereas at HRT longer than optimal, the digester components
are not effectively utilized [51]. If the HRT is short and bacterial
loss exceeds the growth rate of bacteria, wash-out' occurs and the
process can fail [37,52].
As far as algal biomass is considered, HRT below 10 days results
is low methane productivity (Table 5). According to Ras et al. [36],
methane productions from microalgae as a function of time can be
t in inverse exponential curve, characterized by an increase
between 10 and 30 days HRT and reaching a stable level after
30th day. Summing up, when the process is operated at low
loading rate and long hydraulic retention time, methane yield is
constant and maximal [13].

Some bottlenecks are identied to digest microalgae anaerobically. Inhibitory factors cause an adverse shift in the microbial
population or limitation of bacterial growth. Inhibition is often
indicated by a reduction of the steady-state rate of methane
production and accumulation of organic acids [13,38]. In one of
the oldest reports [12] authors mention that amount of destructed
volatile matter and gas produced from algae were always lower
that obtained from raw sewage sludge fermentation. Methane
conversion efciencies of microalgal biomass and raw sewage
sludge reached 36.444.3% and 5760% in case of mesophilic
fermentation, respectively. In the same study authors also report
that digested algae had some undesirable physical characteristics
sludge was highly colloidal, gelatinous and poorly dewatered as
well as had the odor of fresh cow manure. It appears that
microalgal biomass either require more time to degrade or it
cannot further degrade [36]. Some inhibition factors could be
reduced by various operations, e.g. pretreatment of the substrate
or co-digestion with other material. Acclimation of the digester
microbial community to microalgal biomass digestion may also
improve the methane yield [54].

496

E. Kwietniewska, J. Tys / Renewable and Sustainable Energy Reviews 34 (2014) 491500

3.1. Ammonia
Ammonia is an important source of nitrogen for bacteria and
low concentrations of ammonia (below 200 mg l  1) are benecial
to the process [38]. However, it has been found that the specic
activity of methanogenic bacteria decreases with increasing concentrations of ammonia [8]. There are several mechanisms responsible for ammonia inhibition: change in the intracellular pH,
increase of maintenance energy requirement as well as inhibition
of a specic enzyme reaction [38]. Moreover, high ammonia
concentration in the digester decreases the deamination activity
of proteolytic bacteria [55]. Free ammonia seems to be the main
cause of inhibition because it is freely, passively membranepermeable and causes proton imbalance and/or potassium deciency [38]. An increase in pH results in the shift of ionized form
(NH4 ) to free ammonia and increased toxicity. Process instability
due to ammonia often results in volatile fatty acids accumulation,
which leads to a decrease in pH and thereby declining concentration of free ammonia. This interaction may lead to an inhibited
steady state, a condition where the process is running stably but
with a reduced biomethane yield [38]. Higher process temperature
also results in a higher excretion of free ammonia. Chen et al. [38]
reports that decrease in operating temperature from 60 to 37 1C in
anaerobic digesters with a high ammonia concentration provided
an increase in biogas yield.
However, methanogens can adapt to ammonia concentrations
above 1700 mg l  1 without the occurrence of a lag-phase if
ammonium-nitrogen concentration increases slowly [38,56]. In
study of Yang et al. [57] repeated batch fermentation was applied
as a method of adaptation methanogens to high-proteinaceous
algal biomass. Methane production rate was increased from
17 ml CH4 day  1 to 28 ml CH4 day  1, but on the other hand,
ammonia was being accumulated in the fermenter and slightly
lowered methane yield in every subsequent cycle.
Highly proteinaceous composition of algae also contribute to
formation of a digested sludge with very low C/N ratio5 [12].
3.2. Sulfur
Sulfur is an element required for methanogenic bacteria, and
moreover cells of the methanogens contain more sulfur than other
groups of anaerobic organisms [38]. Although, sulfur in form of
sulfate or sulde may become an inhibitor in the anaerobic
digestion process. As sulfate is reduced by sulfate reducing
bacteria, competition for the substrates between sulfate reducing
bacteria and methanogenic bacteria can constitute inhibition.
Furthermore, the inhibition results from the toxicity of sulde
and produced hydrogen sulde to other groups of bacteria. H2S is
toxic because it diffuses into the cytoplasm by cell membranes and
may form disulde cross-links between polypeptide chains and
denaturate the proteins. It was also observed that toxicity of
sulde increases with pH [38]. Sulfur content of microalgal cells
is rather low and ranges between 1.5 and 16 g mg  1 dry weight
[25] which corresponds to 0.151.96% by dry weight [5860].

less active because of mass transfer limitations. Excessive potassium leads to passive inux of K-ions and neutralize the membrane potential [38]. In case of marine algae especially, high
concentration of sodium in their biomass might be an inhibitor
of the anaerobic digestion due to osmotic stress and dehydratation
[13,38]. However, in study of Lakaniemi et al. [61] it turned out
that the inhibiting element of fermentation of marine species
Dunaliella tertiolecta could be rather chloride than sodium. Authors
mention that on freeze-dried biomass of this species, salts on the
surface of the biomass were visible. However, anaerobic microora
is feasible to adapt to salt environment and inhibiting effect may
not occur [13]. Acclimation of methanogens to sodium can shorten
the lag phase before biogas production begins [38].
Ogejo et al. [37] reports that digesters treating municipal
wastewater have failed sometimes because of copper, zinc, chromium, and nickel were present in the substrate. Toxicity of heavy
metals results from that heavy metals are not biodegradable, thus
may accumulate to toxic concentrations and negatively affect
enzyme function [38]. According to Golueke et al. [12], aluminum
in algae harvested by alum-occulation had no apparent inhibitory effect on digester activity, but on the other hand Chen et al.
[38] mentions that aluminum was reported to inhibit the growth
of methanogens and to reduce biogas production and methane
content in the biogas.
3.4. Fatty acids
Short chainvolatile fatty acids are not toxic themselves. They
are produced and used as nutrients normally in an active digester.
However, their inhibiting effects could be indirect as they might
lower the pH to undesirable level [12]. The methanogens will not
be able to metabolise the acetate produced by the acetogenic
organisms until the number of methanogenic organisms has
increased sufciently. This is especially true of feedstocks which
are rapidly hydrolyzed [9]. Increase of VFA content may be caused
by increased activity of the acidogenic bacteria coupled with
inhibition of methanogens. Various acids concentrations are good
indicators of the digester condition. It was demonstrated that
propionic to acetic acid ratio 4 1.4 and acetic acid content of
4800 mg l  1 is a signal of digester failure. The accumulation of
butyric and valeric acid also results in inhibition of the process and
cause digester failure at 46500 mg l  1. The VFA to alkalinity ratio
adequately characterizes the digestion process: the lower ratio, the
higher methane yield is [41].
Long chain fatty acids can also constitute inhibition to anaerobic digestion process. Long chain fatty acids retard gram-positive
bacteria activity, which includes methanogens. Toxicity of long
chain fatty acids results from adsorption onto the cell wall or
membrane, causing disorientation of essential groups on the cell
membrane and thus transport and protection function problems
[38,55]. This factor must be considered as far as microalgal
biomass can reach high content of lipids if cells are cultivated in
stress conditions.
3.5. Cell wall

3.3. Macroelements, microelements, heavy metals

Supplementation of certain metals increases biogas production,
due to the fact that some metals are used as a part of the enzymes
structure of the bacteria [9]. On the other hand, some macroelements, microelements and heavy metals may have toxic effect on
anaerobic microora in digester. High concentrations of essential
alkali metals like magnesium, calcium, sodium, and potassium can
be toxic to anaerobic bacteria [37]. Excessive amounts of calcium
result in precipitation of carbonate and phosphate and cause
scaling of reactors as well as of bacterial cells. Scaled biomass is

Cell wall is considered to be the main characteristics of

digestion capability of algal biomass [6264]. Microscopic analysis
of Golueke et al. [12] showed that a large amount of algal cells in
the sludge, from both thermophilic and mesophilic experiments,
were intact (but not viable) which indicates that probably cell wall
of algae inhibited the microbial penetration. It was also reported
that photosynthetic activity of the intact algae in the digester can
cause the presence of oxygen in the biogas [13], that is why the
digesters should be kept in the darkness when microalgal biomass
is digested [36]. However, Mussgnug et al. [62] notes that even

E. Kwietniewska, J. Tys / Renewable and Sustainable Energy Reviews 34 (2014) 491500

after six months after transfer, there were intact cells of Scenedesmus obliquus present in the digester, which indicates that this
species not only has resistant cell wall but is also probably able to
utilize variety of sugars and organic acids for heterotrophic
growth.
In general, algal cell walls are made up of two components: the
brillar component, which forms the skeleton of the wall (cellulose, mannan, xylans), and the amorphous component, which
forms a matrix within which the brillar component is submerged
[65]. Ultrastructure of the cell walls of genus Chlorella and
Scenedesmus (Chlorococcales) are well studied. Cell walls of these
genera comprise of two layers. The inner layer of the cell wall
consists of cellulose microbrils and amorphous matrix and outer
layer may appear in two formshomogenous or as trilaminar
sheath (TLS) [66]. The trilaminar component includes a substance
considered previously to be the polymerized carotenoid, sporopollenin [67]. Sporopollenin is an UV autouorescent lignin-like
biopolymer surrounding the zygotes of several charophytic algae
species, such as Coleochaete, and constituting the outer wall of
noneuphyllophyte spores and euphyllophyte pollen [68]. Sporopollenin is polymerized from hydroxylated fatty acids and phenolics. Nowadays sporopollenin-like substances in algal cell walls are
called algaenan or acetolysis-resistant biopolymer (ARB). Algaenan
is resistant to extreme extraction procedures including acetolysis
[67] as well as to organic solvents, bases and non-oxidizing acids
[66]. In studies conducted by Gunninson and Alexander [69], cell
walls of the algae were fractionated, and the fractions least
susceptible to microbial degradation were the sporopollenin-like
substance.
On the other hand, Grossi et al. [70] in the study conclude that
presence of algaenan-containing cell wall do not protect other
components of the cell from microbial degradation under either
oxic or anaerobic conditions.
One of the main limits on the anaerobic digestion process, as
far as higher plant are considered, is its inability to degrade lignin
(a major component of wood). This is in contrast with the process
of aerobic biodegradation [6]. The chemical composition and
structure of lignocellulosic materials lowers the rate of biodegradation [8]. Lignin has generally been considered to be a hallmark
of tracheophytes, but there are sporadic reports in the literature
describing the detection of lignin or lignin-like compounds from
nonvascular plants, including brown algae, charophytic algae and
mosses [68]. Also lignin derivatives with aldehyde groups or
apolar substituents may affect methanogens [38]. The most
popular species for cultivation like Chlorella are believed to
produce no lignin and only a little cellulose or other carbohydrate
wall material [65,68]. However, Sui et al. [71] analyzed lignin

497

content in Chlorella cell walls and the results appeared to be

unexpected. Two analysis methods were used and gave values of
2.456.35 wt% (of extract-free cells). Both analyses seem to indicate the presence of phenylpropane units, but that conclusion has
not been conrmed.
3.5.1. Pretreatment
Various physical, chemical and enzymatic pretreatments are
used in order to increase substrate solubility and accelerate the
biodegradation rate of solid organic waste [8]. These methods of
pretreatment can also signicantly and efciently increase the
conversion yield of the algal organic matter into methane by
increasing the accessibility to algal protoplast [13]. There were
some studies concerning pretreatment of microalgal biomass for
anaerobic digestion carried out [63,7275]. A few methods of
pretreatment were examined, e.g. thermal, ultrasound, pressing,
chemical, enzymatic. Most of the results indicate that pretreatment is a crucial step of the whole process and the yields of biogas
and methane are higher after the pretreatment is applied. Subjecting biomass to physicochemical treatment weakens the rigid
cell wall structure and thus allows methanogens to consume the
organic compounds inside the cell. Moreover, smaller particles,
with higher surface area to volume ratios, will have higher
reaction efciency during anaerobic digestion for biogas [20]. Yang
et al. [57] state that two-stage process of hydrogen and methane
production can be a promising idea of energy acquiring as
methane yields are higher compared to regular one-step anaerobic
digestion. Hydrogen production before anaerobic digestion process can play as a kind of pretreatment step for methanogenic
bacteria, because it enhances hydrolysis and improves biodegrability of algal remnants. One of the exceptions is drying process
which decreases fermentative potential of microalgal biomass
[62]. It must be noticed that excessive energy input for pretreatment might negatively impact the economic feasibility of the
whole process of biofuel production [55].
Table 5 shows the results of empirical studies conducted
concerning anaerobic digestion of the whole algal biomass.

4. Co-digestion
Co-digestion is a substrate treatment method in which different
substrates are mixed and treated together. It is also termed as cofermentation. Co-digestion is an energy-efcient process that can
improve fermentation performance by adding a secondary substrate
that supplies nutrients that initial substrate is lacking, improving
physicochemical parameters of the batch [55]. Co-digestion is

Table 6
Summary of the results of empirical studies of co-digestion of microalgal biomass with other substrates.
Species

Co-substrate

C/N

Temp.
[1C]

Methane yield [m3 kg  1 VS

introduced]

HRT References

Spirulina maxima

6.2

35

0.36

20

Spirulina maxima

Primary domestic sewage sludge

49.4%
Peat hydrolyzate 35%

6.3

35

0.28

20

Spirulina maxima

Spent sulte liquor 9.3%

4.7

35

0.25

20

Chlorella post transesteried residues

Chlorella post transesteried residues
Scenedesmus spp. and Chlorella spp.
Post-lipid-extracted residues of Nannochloropsis
salina
Microcystis sp. from Taihu lake
Species unknown
Species unknown

Glycerol
Glycerol
Waste paper 50%
Lipid-rich fat, oil and grease waste
50%
Corn straw
18 ml algae 0.5 ml soybean oil
18 ml algae 0.082 ml glycerin

8.53
12.44
18
-

40
35
35
37

0.308
0.295
1170 ml l  1 day  1
0.54

15
15
10
27

Samson and LeDuy

[44]
Samson and LeDuy
[44]
Samson and LeDuy
[44]
Ehimen et al. [41]
Ehimen et al. [41]
Yen and Brune [45]
Park and Li [55]

20

35
30
30

0.325
1794 ml of biogas
1013 ml of biogas

30
28
28

Zhong et al. [76]

Salerno et al. [54]
Salerno et al. [54]

498

E. Kwietniewska, J. Tys / Renewable and Sustainable Energy Reviews 34 (2014) 491500

preferably used for improving yields of anaerobic digestion due to

its numeral benets. Dilution of toxic compounds, increased load of
biodegradable organic matter, alleviating imbalance of nutrients,
adjustment of the carbon-to-nitrogen (C/N) ratio, preventing inhibition, synergistic effect of microorganisms, easier handling of
mixed substrates, economic advantages due to equipment sharing
and better biogas yield are the potential benets that are achieved
in a co-digestion process [8,9,13,55,76]. Well-maintained nutrient
balance of the fermenter not only gives higher methane yield but
also allows higher loading rates, improving the economic feasibility
of the process [55]. Due to the fact that microalgal biomass has a
very low C/N ratio (46), co-digestion with carbon-rich co-substrate
is widely recommended.
In Samson and LeDuy [44] study, spent sulte liquor (SSL)
turned out to be inappropriate as co-substrate. Despite the high
C/N ratio, the more SSL was introduced to algal biomass, the less
methane was produced or the process failed. Authors did not
explain the possible reasons for such case. Yen and Brune [45]
observed that C/N ratio 11.818.0 is most suitable for anaerobic
digestion of algal sludge with waste paper and the achieved biogas
production is similar in this range of C/N ratio. The authors also
analyzed the cellulase activity in the process, as cellulose hydrolysis is considered the rate-limiting step of the anaerobic digestion.
Co-digestion of algal sludge mixed with waste paper (50%/50%)
resulted in high cellulase activity and highest methane production.
However, digestion of waste paper alone resulted in highest
cellulase activity but poor methane production. Therefore, it is
assumed that algal biomass contributed some key components
that improved activity of methanogens. Simultaneously, increase in
cellulase activity might be benecial for biodegradation of algal cell
walls. Gonzles-Fernndez et al. [77] co-digested microalgal biomass with swine manure. However, anaerobic digestion of algae as
a sole substrate as well as algae as a predominant co-substrate
resulted
in
low
methane
productivity
(128.9
143.0 mL CH4 g COD  1) and the highest methane yields were
achieved when swine manure was fermented as a sole substrate
(245.9 ml CH4 g COD  1). Microscopic analysis proved that algal
species used in the study were mainly C. vulgaris and S. obliquus.
These species are known to have a recalcitrant cell wall and hence it
was assumed that the cell wall was responsible for the inhibition of
biogas production. In study conducted by Zhong et al. [76], the
highest biogas and methane yield was reached by co-digestion of
blue algae biomass with corn straw at C/N ratio adjusted to 20. At
such C/N ratio, volatile solids reduction (%) and methane content in
biogas (%) was also the highest. Increase of methane productivity by
61.69% was noted in comparison to digestion of algal biomass solely.
The authors emphasize, that positive synergistic effect occurred due
to well-balanced nutrients, increased buffering capacity and probable increased cellulose activity, as a result of co-digestion. Salerno
et al. [54] noticed that at rst two to three weeks of the experiment
concerning co-digestion with soybean oil, kind of inhibition or
acclimation occurred and after this period biogas production rate
increased abruptly. The same situation appeared when twofold
amount of algae was digested. Authors supposed that inhibition
occurred due to algal biomass excess.
The results of experiments of co-digestion are presented in
Table 6.

5. Anaerobic digestion of algal remnants

Anaerobic digestion can be also effectively used as a means of
producing biomethane from remnants which develop after conversion (e.g. extraction) of algal feedstock into fuel [20]. For example,
the biodiesel production process results in the co-production of
biomass residues and glycerol [41]. As microalgal-derived biodiesel
appears to be a promising alternative to petroleum-based liquid fuel,
the utilization or disposal of post-extraction residues is a key issue
considering balancing both energetic and economic aspects of algal
derived biofuel industry commercialization. Post-extraction algal
remnants accounts for approximately 65% of the harvested biomass
and thus it can generate additional energy through methane
fermentation. By integrating anaerobic digestion with algal biodiesel
production, whole harvested biomass can be utilized and efciency
can be increased [55]. The economic value of the produced methane
is equivalent to about $100 per ton of digested biomass, which is
signicant in terms of reducing the overall cost of liquid biofuels
production [20].
When lipids are extracted from algal biomass before digestion,
the potential biogas yield is lower and ammonium release is higher,
because of higher protein content by percent. The high ammonium
concentration may then strongly limit and even jeopardize the
process stability. To manage this rich nitrogen substrate, a codigestion with a poor nitrogen substrate is thus necessary [13]. An
example of such substrate might be another lipid-rich substrates
and waste, which offset the C/N ratio imbalance. However, lipids are
characterized by low alkalinity and buffering capacity which makes
them vulnerable to inhibition. Low alkalinity can be offset by
increasing protein content, thus lipid degradation can be also
increased due to favorable alkalinity [55].
In the study of Ehimen et al. [41] algal residues were co-digested
with glycerol. Thanks to the co-digestion the C/N ratio was increased
and simultaneously the CH4 yield was increased by 450%, although,
authors recorded no improvement of biogas productivity with C/N
ratio higher than 12.44. In all cases, it was observed that an increase in
the digestion time with a corresponding reduction in the loading rates,
led to increased CH4 yields. Unexpected results were achieved by Park
and Li [55]. Co-digestion of 50% post-lipid-extracted algal biomass
with lipid-rich fat, oil and grease waste gave very high methane yield
of 0.54 m3 kg  1 VS. Calculations prove that this result is 23% greater
than the theoretical methane potential which indicates that synergetic
effect caused by optimal ratio of the substrates occurred.
However, lipid extraction of biomass containing less than 40%
of lipids combined with anaerobic digestion of the remnants is not
effective in terms of energy nor in term of the costs. When the cell
lipid content does not exceed 40%, anaerobic digestion of the
whole biomass appears to be the optimal strategy on an energy
balance basis, for the energetic recovery of cell biomass [13].
In Table 7 the results of empirical studies concerning anaerobic
digestion of algal remnants are presented.

6. Future perspectives
Anaerobic digestion of various organic wastes works well e.g. in
Germany which is undisputed leader in biogas production in

Table 7
Summary of the results of empirical studies of anaerobic digestion of algal remnants.
Species

C/N

Temp. [1C]

Methane yield [m3 kg  1 VS introduced]

HRT

References

Post-lipid-extracted residues of Nannochloropsis salina

Chlorella post transesteried residues
Lipid-extracted microalgal biomass residues from Scenedesmus

5.4
10.8

37
35
37

0.13
0.245
0.323

40
15
50

Park and Li [55]

Ehimen et al. [41]
Yang et al. [57]

E. Kwietniewska, J. Tys / Renewable and Sustainable Energy Reviews 34 (2014) 491500

Europe [78] or in Sweden which is famous of biogas-supplied city

buses. The technology is well known and functions successfully in
industry for years. Future research in this eld will probably
include development of new methods of process monitoring and
control (real time measurements), which are lacking at present;
improvement of pretreatment and co-digestion methods; and also
molecular analyses of microbial community in order to enhance
degradation process. There are some studies on immobilization of
bacteria as a way to improve reactor efciency and yields as well
as to reduce HRT and possibility of reactor failure. Modernizing the
design of plants, in particular mixing systems, is also a vital aspect,
as mixing is important for the access of microorganisms to the
substrate [8,9,79].
Future perspectives of anaerobic digestion of microalgal biomass are inseparable from cost-effectiveness of the process on
industrial scale. Many reports and analyses based on prevailing
state-of-the-art technologies and engineering indicate that such
system would not be cost-effective. Electric consumption and,
associated with it, environmental impact are the main bottlenecks
to be overcome. The main trouble spot are immature technologies
used for growing and harvesting of algal cells [80]. Particularly,
high cost of building and maintaining photobioreactors or ponds
and low density of cultures are the weakest points. Improving of
pretreatment methods is also a crucial aspect regarding microalgal
biomass digestion as many publications report the low bioavailability of resistant compounds of the cells. There is a chance for
solving this issue in selection of algal species for cultivation which
does not have a cell wall but simultaneously has high growth rate
and good biomass composition [36,64]. It is also reported, that
viability and sustainability of the process would rise if biogas
production from microalgae was coupled with prior biodiesel
recovery [13,80,81] or any other valuable product (e.g. pharmaceutics). The solution that may also reduce unfavorable economic
and ecological impact is to connect other alternate energy acquiring installations to the whole system, like solar panels or wind
turbines [80]. Finally, there is a strong need to develop detailed
models and carry out technoeconomic analyses and life-cycle
assessments based on various versions and options of the technology in order to discover strong and weak points of the process and
suggest advanced, innovative solutions.

7. Conclusions
As it was shown in a few empirical studies, anaerobic digestion
of microalgal biomass is possible and gives quite good methane
yields. Despite these pleasing results, the process of methane
fermentation of microalgae is still not well examined. Many
studies are described supercially, with only few aspects of the
whole process taken into consideration. Partially, this gap is
caused by the fact that cultivation of microalgal biomass is not
cost-effective for the time being. Nevertheless, mainly due to its
high growth rate, biomass of microalgae is a realistic, alternative
biomass feedstock of the future. This indicates that anaerobic
digestion of microalgae should be studied more carefully and in
detail.

Acknowledgements
This work was nanced from the project Development of
physiological-technical assumptions for the production of algae for
purposes of energy generation No. N N313 705940, Ministry of
Science and Higher Education, Poland.

499

References
[1] U.S. Energy Information Administration. International Energy Outlook; 2011.
Washington, DC: DOE/EIA-0484(2011).
[2] Cornelissen S, Koper M, Deng YY. The role of bioenergy in a fully sustainable
global energy system. Biomass Bioenergy 2012;41:2133.
[3] Appels L, Lauwers J, Degrve J, Helsen L, Lievens B, Willems K, et al. Anaerobic
digestion in global bio-energy production: potential and research challenges.
Renewable Sustainable Energy Rev 2011;15:4295301.
[4] Vergara-Fernandez A, Vargas G, Alarcon N, Velasco A. Evaluation of marine
algae as a source of biogas in a two-stage anaerobic reactor system. Biomass
Bioenergy 2008;32:33844.
[5] Brennan L, Owende P. Biofuels from microalgaea review of technologies for
production, processing, and extractions of biofuels and co-products. Renewable Sustainable Energy Rev 2010;14(2):55777.
[6] European Commission. Reference document on best available techniques for
the waste treatments industries. Integrated pollution prevention and control
2005.
[7] Karagiannidis A, Perkoulidis G. A multi-criteria ranking of different technologies for the anaerobic digestion for energy recovery of the organic fraction of
municipal solid wastes. Bioresour Technol 2009;100:235560.
[8] Khalid A, Arshad M, Anjum M, Mahmood T, Dawson L. Reviewthe anaerobic
digestion of solid organic waste. Waste Manage 2011;31:173744.
[9] Ward AJ, Hobbs PJ, Holliman PJ, Jones DL. Optimisation of the anaerobic
digestion of agricultural resources. Bioresour Technol 2008;99:792840.
[10] Singh J, Gu S. Commercialization potential of microalgae for biofuels production. Renewable Sustainable Energy Rev 2010;14:2596610.
[11] Miyamoto K. Renewable biological systems for alternative sustainable energy
production. FAO agricultural services bulletin 1997;128.
[12] Golueke CG, Oswald WJ, Gotaas HB. Anaerobic digestion of algae. Appl
Microbiol 1957;5(1):4755.
[13] Sialve B, Bernet N, Bernard O. Anaerobic digestion of microalgae as a necessary
step to make microalgal biodiesel sustainable. Biotechnol Adv 2009;27:
40916.
[14] Barber RD, Ferry JG. Methanogenesis. Encyclopedia of life sciences. London:
Nature Publishing Group; 2001.
[15] Bitton G. Wastewater microbiology. Wiley series in ecological and applied
microbiology. 3rd ed.. Hoboken: New Jersey: John Wiley & Sons, Inc.; 2005.
[16] Samson R, LeDuy A. Biogas production from anaerobic digestion of Spirulina
maxima algal biomass. Biotechnol Bioeng 1982;24:191924.
[17] Pulz O, Gross W. Valuable products from biotechnology of microalgae. Appl
Microbiol Biotechnol 2004;65:63548.
[18] Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25:294306.
[19] Wijffels RH, Barbosa MJ. An outlook on microalgal biofuels. Science
2010;329:7969.
[20] US DOE. National algal biofuels technology roadmap. U.S. Department of
Energy, Ofce of Energy Efciency and Renewable Energy. Biomass Program
2010.
[21] Heredia-Arroyo T, Wei W, Hu B. Oil accumulation via heterotrophic/mixotrophic Chlorella protothecoides. Appl Biochem Biotechnol 2010;162:197895.
[22] Ruangsomboon S. Effect of light, nutrient, cultivation time and salinity on lipid
production of newly isolated strain of the green microalga, Botryococcus
braunii KMITL 2. Bioresour Technol 2012;109:2615.
[23] Hu Q. Environmental effects on cell composition. In: Richmond A, editor.
Handbook of microalgal culture: biotechnology and applied phycology.
Victoria: Blackwell Publishing; 2004. p. 8393.
[24] Illman AM, Scragg AH, Shales SW. Increase in Chlorella strains caloric values
when grown in low nitrogen medium. Enzyme Microb Technol
2000;27:6315.
[25] Grobbelaar JU. Algal nutritionmineral nutrition. In: Richmond A, editor.
Handbook of microalgal culture: biotechnology and applied phycology.
Victoria: Blackwell Publishing; 2004. p. 97115.
[26] Moody L, Burns R, Wu-Haan W, Spaji R. Use of biochemical methane
potential (BMP) assays for predicting and enhancing anaerobic digester
performance. In: Agricultural engineering; 2009. 44th Croatian and fourth
international symposium on agriculture, p. 930934.
[27] Angelidaki I, Sanders W. Assessment of the anaerobic biodegradability of
macropollutants. Rev Environ Sci Biotechnol 2004;3:11729.
[28] van Haandel AC. van der Lubbe JGM. Handbook biological wastewater
treatmentdesign of activated sludge systems. Leidschendam, The Netherlands: Quist Publishing; 2007.
[29] Heaven S, Milledge J, Zhang Y. Comments on Anaerobic digestion of microalgae as a necessary step to make microalgal biodiesel sustainable. Biotechnol
Adv 2011;29(1):1647.
[30] CimochowiczRybicka M. Effect of anaerobic sludge composition on a biogas
production. In: Plaza E, Levlin E, Hultman B, editors. Sustainable municipal
sludge and solid waste handling, Report no 7. Trita-Ami report 3073; 2000. p.
6976.
[31] Lauwers J, Appels L, Thompson IP, Degrve J, Van Impe JF, Dewil R.
Mathematical modelling of anaerobic digestion of biomass and waste: power
and limitations. Prog Energy Combust Sci 2013;39:383402.
[32] Gavala HN, Angelidaki I, Ahring BK. Kinetics and modeling of anaerobic
digestion process. In: Ahring BK, editor. Biomethanation I. Adv Biochem
Engin/Biotechnol, 81. BerlinHeidelberg: Springer-Verlag; 2003. p. 5793.

500

E. Kwietniewska, J. Tys / Renewable and Sustainable Energy Reviews 34 (2014) 491500

[33] Batstone DJ, Keller J, Angelidaki I, Kalyuzhnyi SV, Pavlostathis SG, Rozzi A, et al.
IWA task group on modeling of anaerobic digestion processes. Anaerobic
digestion model no. 1 (ADM1). London: IWA Publishing; 2002.
[34] Batstone DJ, Keller J, Angelidaki I, Kalyuzhnyi SV, Pavlostathis SG, Rozzi A, et al.
The IWA anaerobic digestion model no 1 (ADM1). Water Sci Technol 2002;45
(10):6573.
[35] Mairet F, Bernard O, Ras M, Lardon L, Steyer JP. Modeling anaerobic digestion
of microalgae using ADM1. Bioresour Technol 2011;102:68239.
[36] Ras M, Lardon L, Bruno S, Bernet N, Steyer JP. Experimental study on a coupled
process of production and anaerobic digestion of Chlorella vulgaris. Bioresour
Technol 2011;102(1):2006.
[37] Ogejo JA, Wen Z, Ignosh J, Bendfeldt E, Collins ER. Biomethane technology.
Virginia Cooperative Extencion. Publication; 2009; 442881.
[38] Chen Y, Cheng JJ, Creamer KS. Inhibition of anaerobic digestion process: a
review. Bioresour Technol 2008;99:404464.
[39] Chae KJ, Jang A, Yim SK, Kim IS. The effects of digestion temperature and
temperature shock on the biogas yields from the mesophilic anaerobic
digestion of swine manure. Bioresour Technol 2008;99:16.
[40] Appels L, Baeyens J, Degreve J, Dewil R. Principles and potential of the
anaerobic digestion of waste-activated sludge. Prog Energy Combust
2008;34:75581.
[41] Ehimen EA, Sun ZF, Carrington CG, Bircj EJ, Eaton-Rye JJ. Anaerobic digestion of
microalgae residues resulting from the biodiesel production process. Appl
Energy 2011;88:345463.
[42] Shelef G, Sukenik A, Green M. Microalgae harvesting and processing: a
literature review. A subcontract report. Solar Energy Research Institute.
Prepared for the U.S. Department of Energy. Haifa, Israel: Technion Research
and Development Foundation ltd; 1984.
[43] Patil PD, Gude VG, Mannarswamy A, Deng S, Cooke P, Munson-McGee S, et al.
Optimization of direct conversion of wet algae to biodiesel under supercritical
methanol conditions. Bioresour Technol 2011;102:11822.
[44] Samson R, LeDuy A. Improved performance of anaerobic digestion of Spirulina
maxima algal biomass by addition of carbon-rich wastes. Biotechnol Lett
1983;5(10):67782.
[45] Yen HW, Brune DE. Anaerobic co-digestion of algal sludge and waste paper to
produce methane. Bioresour Technol 2007;98:1304.
[46] Rincn B, Travieso L, Snchez E, de los Angeles Martn M, Martn A, Raposo F,
et al. The effect of organic loading rate on the anaerobic digestion of twophase olive mill solid residue derived from fruits with low ripening index. J
Chem Technol Biotechnol 2007;82:25966.
[47] Babaee A, Shayegan J. Effect of organic loading rates (OLR) on production of
methane from anaerobic digestion of vegetables waste. World renewable
energy congress. Sweden: Linkping; 2011 (8-13 May).
[48] De Schamphelaire L, Verstraete W. Revival of the biological sunlight-to-biogas
energy conversion system. Biotechnol Bioeng 2009;103(2):296304.
[49] Monnet F. An introduction to anaerobic digestion of organic wastesnal
report. Remade Scotland 2003.
[50] Ekama GA, Wentzel MC. Organic material removal. In: Henze M, van
Loosdrecht, Ekama G, Brdjanovic D, editors. Biological wastewater treatment:
principles, modeling, and design. IWA Publishing, Cambridge University Press;
2008.
[51] Nagamani B, Ramasamy K. Biogas production technology: an Indian perspective. Curr Sci India 1999;77(1):4455.
[52] Burke DA. Dairy waste anaerobic digestion handbookoptions for recovering
benecial products from dairy manure. Environ Energy Company; 2001.
[53] Zeng S, Yuan X, Shi X, Qiu Y. Effect of inoculum/substrate ratio on methane
yield and orthophosphate release from anaerobic digestion of Microcystis spp.
J Hazard Mater 2010;178(1-3):8993.
[54] SalernoM, Nurdogan Y, Lundquist TJ. Biogas production from algae biomass
harvested at wastewater treatment ponds. In: Bioenergy engineering conference sponsored by ASABE Hyatt regency, Seattle, Washington October 11
14; 2009.
[55] Park S, Li Y. Evaluation of methane production and macronutrient degradation
in the anaerobic co-digestion of algae biomass residue and lipid waste.
Bioresour Technol 2012;111:428.

[56] Koster IW, Lettinga G. The inuence of ammonium-nitrogen on the specic

activity of pelletized methanogenic sludge. Agr Wastes 1984;9(3):20516.
[57] Yang Z, Guo R, Xu X, Fan X, Luo S. Hydrogen and methane production from
lipid-extracted microalgal biomass residues. Int J Hydrogen Energy
2011;36:346570.
[58] Friis JC, Holm C, Halling-Serensen B. Evaluation of elemental composition of
algal biomass as toxical endpoint. Chemosphere 1998;37(13):266576.
[59] Mandalam RK, Palsson OP. Elemental balancing of biomass and medium
composition enhances growth capacity in high-density Chlorella vulgaris
cultures. Biotechnol Bioeng 1998;59(5):60511.
[60] Barsanti L, Gualtieri P. Algae: anatomy, biochemistry, and biotechnology. Boca
Raton: CRC Press; 2006.
[61] Lakaniemi AM, Hulatt CJ, Thomas DN, Tuovinen OH, Puhakka JA. Biogenic
hydrogen and methane production from Chlorella vulgaris and Dunaliella
tertiolecta biomass. Biotechnol Biofuels 2011;4:34.
[62] Mussgnug JH, Klassen V, Schlter A, Kruse O. Microalgae as substrates for
fermentative biogas production in a combined biorenery concept. J Biotechnol 2010;150:516.
[63] Gonzles-Fernndez C, Sialve B, Bernet N, Steyer JP. Thermal pretreatment to
improve methane production of Scenedesmus biomass. Biomass Bioenergy
2012;40:10511.
[64] Zamalloa C, Boon N, Verstraete W. Anaerobic digestibility of Scenedesmus
obliquus and Phaeodactylum tricornutum under mesophilic and thermophilic
conditions. Appl. Energy 2012;92:7338.
[65] Lee RE. Phycology. 4th ed.Cambridge University Press; 2008.
[66] Burczyk J. Studies concerning carotenoids and sporopollenin in algal cell walls.
Postdoctoral degree thesis. Cracov; Institute of Zootechnics; 1982. (in Polish).
[67] AWJr Atkinson, BES Gunning, PCL. John. Sporopollenin in the cell wall of
Chlorella and other algae: ultrastructure, chemistry, and incorporation of 14Cacetate, studied in synchronous sultures. Planta 1972;107:132.
[68] Weng JK, Chapple C. The origin and evolution of lignin biosynthesis. New
Phytol 2010;187:27385.
[69] Gunninson D, Alexander M. Basis for the resistance of several algae to
microbial decomposition. Appl Microbiol 1975;29(6):72938.
[70] Grossi V, Blokker P, Sinninghe Damst JS. Anaerobic biodegradation of lipids of
the marine microalga Nannochloropsis salina. Org Geochem 2001;32:795808.
[71] Sui Z, Gizaw Y, BeMiller JN. Extraction of polysaccharides from a species of
Chlorella. Carbohydr Polym 2012;90:17.
[72] Mayo MH, Nicholson AD, Hansen LD, Hansen JC. Chemical treatment of algae
to facilitate biogas production by anaerobic digestion. Trans ASABE 2011;54
(4):154750.
[73] Schwede S, Kowalczyk A, Gerber M, Span R. Inuence of different cell
disruption techniques on mono digestion of algal biomass. World renewable
energy congress, 8-13.05. Sweden: Linkping; 2011.
[74] Gonzles-Fernndez C, Sialve B, Bernet N, Steyer JP. Comparison of ultrasound
and thermal pretreatment of Scenedesmus biomass on methane production.
Bioresour Technol 2012;110:6106.
[75] Gerken HG, Donohoe B, Knoshaug EP. Enzymatic cell wall degradation of
Chlorella vulgaris and other microalgae for biofuels production. Planta
2013;237:23953.
[76] Zhong W, Zhang Z, Luo Y, Qiao W, Xiao M, Zhang M. Biogas productivity by codigesting Taihu blue algae with corn straw as an external carbon source.
Bioresour Technol 2012;114:2816.
[77] Gonzles-Fernndez C, Molinuevo-Salces B, Garca-Gonzles MC. Evaluation of
anaerobic codigestion of microalgal biomass and swine manure via response
surface methodology. Appl Energy 2011;88:344853.
[78] Eurobserv ER. Biogas Barometer 2012:6679.
[79] Weiland P. Biogas production: current state and perspectives. Appl Microbiol
Biotechnol 2010;85:84960.
[80] Collet P, Hlias A, Lardon L, Ras M, Goy RA, Steyer JP. Life-cycle assessment of
microalgae culture coupled to biogas production. Bioresour Technol
2011;102:20714.
[81] Harun R, Davidson M, Doyle M, Gopiraj R, Danquah M, Forde G. Technoeconomic analysis of an integrated microalgae photobioreactor, biodiesel and
biogas production facility. Biomass Bioenergy 2011;35:7417.